Cryopreservation of oocytes is becoming crucial for fertility preservation in women. Cancer patients who are about to lose their ovarian function because of cancer treatments, like chemotherapy and radiotherapy, can preserve their undamaged oocytes for later use. Women who desire to have a child at older ages due to their career or marital status can do so by using their preserved young oocytes that are likely to have intact chromosomes. In the past several years, a number of babies were born that were derived from cryopreserved oocytes. Nonetheless, oocyte cryopreservation is still considered as an experimental procedure, and success rates are variable among clinics. Thus, the process of vitrification needs to be further investigated for its safety and robustness by examining it carefully at the cellular and molecular levels. Such approaches are particularly critical to predict any potential damage to oocytes, which may affect their development into healthy individuals. The focus of this proposal is on the mechanisms of meiotic spindle regeneration during vitrification, which utilizes the fast-freezing method to cryopreserve oocytes. The meiotic spindle is a crucial apparatus to segregate the correct number of chromosomes into an oocyte. Damages to the spindle potentially result in chromosome nondisjunction, which is a major cause of developmental failure and birth defects. It is known that meiotic spindle degenerates during oocyte freezing but it regenerates during thawing and warming. However, how the spindle regenerates and whether chromosome alignment is stable without the spindle have not been fully investigated. In that respect, I am pursuing the following three Specific Aims, using the mouse as well as human oocytes. In Specific Aim 1, the localization of several spindle-nucleating components, such as g-tubulin, pericentrin, and NEDD1, will be examined during the vitrification process. My hypothesis is that these nucleating components are undisturbed during oocyte freezing so that they can serve as the organizing center for spindle regeneration after thawing. In Specific Aim 2, I will monitor the dynamic process of spindle regeneration in real time. The regeneration process will be examined by fluorescence time-lapse cinematography using live oocytes that express Green Fluorescent Protein-tagged b-tubulin. This analysis will generate insight into the mode of spindle regeneration in a spatial and temporal manner. In Specific Aim 3, the stability of metaphase chromosomes in the absence of spindle will be investigated. Because oocyte vitrification causes depolymerization of the meiotic spindle, it may destabilize alignment of chromosomes and increase the chance of nondisjunction. I will determine the effect of various vitrification conditions, particularly of the initial equilibration step, on chromosome alignment at the metaphase plate. This information will help us improve the vitrification protocol to establish a more consistent and safer cryopreservation of oocytes.